The major long term objective of this research is to examine the importance of calcium regulated actin filament assembly in the activation of human platelet function. The immediate goal is to characterize biochemically a complex of proteins which may be involved in a calcium mediated regulation of filament assembly which we have recently isolated from human platelet extracts. This complex, which consists of actin and two other polypeptides, binds to actin-DNAase I-agarose only if calcium is available. Addition of the isolate complex to muscle G-actin modulates the assembly of actin filaments if micromolar concentrations of calcium ion are present. The specific aims of the proposal are to: 1) Further purify the complex, separate the individual components and determine subunit structure. 2) Investigate the calcium regulated interactions of the complex and the individual components of the complex with actin and actin filaments. 3) Study the calcium binding properties of the properties of the complex and its components and correlate binding with the inhibition of actin assembly. 4) Look for the presence of the components of the complex in cell types other than platelets using actin-DNAase I-affinity chromatography and specific antibodies. The eventual aim here would be to localize the non-actin components of the complex using immunofluorescence techniques, quantitate them using radioimmunoassays and look at intracellular function by microinjection of antibodies to the components of the complex. 5) Compare the components of the complex with profilin and gelsolin, two molecules which have been implicated in the regulation of actin filaments, and also with calmodulin, a calcium binding protein involved in the calcium mediated regulation of several cellular functions.